Cancer cells often have defects in programmed cell death and do not undergo apoptosis in response to cytotoxic agents. Tumor Necrosis Factor alpha (TNF) and the related cytokines FasL and TRAIL induce apoptosis by similar mechanisms dependent upon caspase activation which often stimulates concomitant disassembly of mitochondria. The proposed studies are designed to examine cellular resistance to apoptosis with a focus on death induced by cytolytic cytokines. Therefore, data from these investigations should have import to the means by which cancer cells evade elimination. It is anticipated that the information gained from these studies will be used to circumvent resistance of cancer cells to apoptosis and thereby increase apoptotic effects of anticancer therapy. TIP-B1 is a novel protein which, when incubated with normally TNF-sensitive target cells (including MCF7 breast cancer cells and normal human fibroblasts), protects them from TNF-induced apoptosis (Berleth et al, 1999, Cancer Research 59:5497-5506 and Berleth et al, 2001 Journal of Leukocyte Biology 69:995-1005). Several "trivial" mechanisms for TIP-B1 protection have been excluded. Thus, TIP-B1 does not proteolyze TNF, nor bind and neutralize TNF, nor interfere with TNF binding to the TNF receptors. TIP-B 1 prevents TNF-induced breakdown of mitochondrial membrane permeability (delta/psi/MM). The overall goal of the proposed studies is to determine the molecular mechanism by which TIP-B1 inhibits TNF-induced apoptosis with a focus on documented TIP-B1 effects on delta/psi/M. Aim 1 examines the initial steps of TIP-BI's mechanism to verify that, consistent with its ability to act exogenously, TIP-B1 is a secreted protein (using western blot analysis) and binds to a specific cellular receptor or transporter (using Scatchard analysis). Aim 2 focuses on TIP-B1 inhibition of delta/psi/M. These studies will examine the effect of TIP-B1 on TNF-induced changes in factors that mediate delta/psi/M (e.g. Bid cleavage and mitochondrial association, caspase 8 activation, reloealization of Bcl-2 family proteins) to ascertain which is the first step in the TNF-induced apoptotic pathway inhibited by TIP-B1. Western blots will examine 1) changes in subcellular location (truncated Bid, BcI2 family proteins) and 2) activation via proteolysis (Bid, caspase 8). Aim 3 investigates if TIP-B1 protects cells from apoptosis induced by FasL and TRAIL and, if so, whether or not this is by the same mechanism by which TIP-B1 prevents TNF-induced apoptosis. Comparison will be made to TIP-BI's lack of protection against doxorubicin-induced apoptosis. Aim 4 examines if decreased TIP-B1 expression mediated by TIP-B 1-targeted siRNAs results in decreased resistance (i.e. increased TNF sensitivity).